Polysaccharides are polymeric carbohydrate structures, formed of repeating units of monosaccharides, joined together by glycosidic bonds. Depending on their chemical composition, polysaccharides are further divided into polysaccharides sensu strictu, which contain only hydroxyl and acetyl groups and aminopolysaccharides, which contain also nitrogen (amino or amido-groups). Natural aminopolysaccharides include chitin and chitosan (only containing hydroxyl, amino and acetyl groups) and keratin sulphate, hyaluronic acid, chondroitin, dermatan sulphates and heparin, which contain also carboxyl and sulphate groups.
Chitin is the most abundant natural aminopolysaccharide and is widely distributed amongst invertebrates including arthropods, nematodes, crustaceans, fungi and some protozoa. Chitin is a polymer of N-acetyl-D-glucosamine. The major form of chitin is α-chitin, as encountered in fungi and arthropods and is characterized by an anti-parallel joining of the polysaccharide chains. The β-form, in which the chains are joined in a parallel way, is rather rare and is found in diatoms and some protists. Chitosan is the N-deacetylated derivative of chitin, although this N-deacetylation is almost never complete. Chitin and chitosan correspond to a family of polymers varying in acetyl content, wherein the degree of acetylation determines whether the aminopolysaccharide is named chitin (degree of acetylation >70%) or chitosan (degree of acetylation <70%).
Chitin is the second most abundant biopolymer in nature after cellulose and, together with its derivatives, it has applications in a wide variety of fields, including medical, pharmaceutical, cosmetics, biotechnology, food industry, agriculture and environmental protection. Despite its huge annual production, chitin still remains an underutilized biomass resource, primarily because of its intractable bulk structure. Therefore, the determination of the concentration of chitinous polysaccharides, as well as the identification of their structure and possible modifications is extremely important, especially for efficient industrial processing. Moreover, it may be important to target specific chitinous polysaccharides, for removal out of the matrix or for modification of their structure.
Special attention has been paid to chitin-binding proteins for detection and purification applications. Chitin-binding proteins are rather common and form a highly diverse group, including but not limited to chitinases, hydrolyzing the internal β-1,4-glycosidic linkages of chitin. Chitin-binding proteins have been detected in bacteria (Folders et al., 2000; Joshi et al., 2008), plants (Iseli et al., 1993), invertebrates (Suetake et al, 2000) and vertebrates (Boot et al., 1995). Chitin-binding proteins are characterized by one or more chitin-binding domains; these binding domains may or may not be linked to a catalytic domain. The binding domains can be isolated and fused to other polypeptides, to create novel chitin-binding proteins.
Chitin-binding domains and chitin-binding proteins do have multiple possible applications: as chitin is absent in vertebrates and plants, chitin-binding domains can be used to detect infection or contamination by chitin-containing organisms, as disclosed in WO 9217786 or WO 2005005955. By fusing a chitin-binding domain to a protein of interest, the protein of interest can be purified on a chitin carrier, using affinity chromatography. Moreover, WO 9411511 discloses biocidal chitin-binding proteins that exert an antifungal activity and can be used as anti-microbial agent. Joshi et al. (2008) describe a chitin-binding protein with insecticidal activity.
However, notwithstanding their possible value, the use of the chitin-binding domains and chitin-binding proteins is rather limited, due to several drawbacks. Most of the chitin-binding domains show cross reactivity with other polysaccharides, limiting the value of the binding domain for specific detection of chitin (Itoh et al., 2002; Guillen et al., 2010). Several chitin-binding domains bind chitin with rather low affinity (Neeraja et al., 2010b), limiting the applications in all fields. Moreover, chitin-binding domains may bind chitin in an irreversible way (Xu et al., 2000; WO 03074660), complicating the use in affinity purification, because the protein cannot be eluted under non-denaturing conditions.
To solve the problems, the introduction of mutations in the chitin-binding domains has been proposed to modulate the chitin-binding activity and to create modified chitin-binding domains with reversible binding properties (Ferrandon et al., 2003; WO 03074660). However, there is still a need for better chitin-binding proteins.
Antibodies are known for their high affinity and specificity. However, production of antibodies against polysaccharides is far from evident, as polysaccharides are hardly immunogenic. Anti-chitin IgA type antibodies have been detected in serum of Crohn's disease, ulcerative colitis and inflammatory bowel disease (WO 2009069007; Dotan et al., 2006; Seow et al., 2008; Seow et al., 2009) and after Candida albicans infection (Sendid et al., 2008). Sales et al. (2001) and Martin et al. (2007) describe the generation of polyclonal rabbit anti-chitin antibodies; U.S. Pat. No. 5,004,699 discloses the use of a mouse serum containing polyclonal anti-chitin antibodies for the detection of fungi and yeasts. However, for the intended uses, monoclonal antibodies, and preferably single chain antibodies are needed. Anti-chitin single chain antibodies have not been disclosed in the art.
WO 94004678 describes immunoglobulins devoid of light chains. It is demonstrated that such antigen-binding proteins comprising an amino acid sequence that comprises four framework regions (FR) and three complementarity-determining regions (CDR), and more specifically VHH, display superior characteristics over monoclonal antibodies as they are extremely stable and retain binding capacity to the target antigen under high temperature (van der Linden et al., 1999), or denaturing conditions (Dolk et al., 2005) and are resistant to harsh regenerating conditions (Saerens et al., 2005). Therefore, the antigen-binding proteins are particularly well suited to be used in industrial processes. However, up to now, such antigen-binding proteins capable of binding polysaccharide are not described, although attempts to make such anti-bodies have been made. Indeed, WO 94004678 disclosed camelid antibodies against carbohydrates, but those are directed against the variant surface antigen of Trypanosoma evansi, which is a glycoprotein. WO 94004678 is neither disclosing nor suggesting antibodies against polysaccharides sensu strictu, or against aminopolysaccharides. Moreover, when De Simone et al. (2008) analyze the immune response in llamas immunized with different types of antigens, i.e., protein, conjugated hapten or polysaccharide (dextran sulphate), no anti-dextran immune response could be detected in the immunized animals, in contrast to clear immune responses to the protein and conjugated hapten antigens; whereas it is relatively easy to generate classical anti-dextran antibodies (Cisar et al., 1975; Bona, 1993). The lack of antibody response in the immunoglobulins devoid of light chains is not unexpected: indeed anti-polysaccharide responses in humans are clearly dominated by IgM and IgG1 types (Bona, 1993) whereas heavy chain antibodies from camelids belong to the IgG2 and IgG3 classes (Hamers-Casterman et al., 1993; Daley et al., 2010). Moreover, it is known that interactions between polysaccharides and individual binding sites in a protein are typically weak and binding strength and specificity is enhanced through polymeric interactions between polysaccharides and oligomeric polysaccharide-binding proteins (Mammen et al., 1998). Being strictly monomeric binders by nature (Muyldermans et al., 2001), VHH are in this respect not well suited to bind polysaccharides. Therefore, the person skilled in the art would assume that it is extremely difficult, if not impossible to raise immunoglobulins devoid of light chains against chitinous polysaccharides. Another complicating factor is the low water-solubility of chitinous polysaccharides, particularly chitin, which means that many standard techniques used for isolating antibodies that are carried out in aqueous solution, cannot be applied.
To obtain and isolate antigen-binding proteins specific for chitinous polysaccharides, an original and innovative approach was used. By immunizing llamas with a complex mixture containing chitinous polysaccharides, rather than with a purified antigen, followed by selecting antigen-binding proteins using immobilized solubilized chitin and finally screening with chitin, prepared directly on a solid surface, we were capable of isolating antigen-binding proteins, more specifically, antigen-binding proteins comprising an amino acid sequence that comprises four framework regions (FR) and three complementarity-determining regions (CDR), wherein the antigen-binding proteins are capable to bind chitinous polysaccharides. Preferably, the antigen-binding proteins are binding to chitin.